Molecular Biology II Eukaryotic Gene-Specific Transcription Factors

Additional factors are required to gain more control over the stimulation of transcription than basal
transcription alone

In addition to promoters, eukaryotic genes contain large regions that act as binding sites for gene-
specific TFs
The sequence-specific binding of gene-specific TFs allows genes to achieve and maintain
controlled levels of tissue-specific expression patterns

Gene-Specific Transcription Factors:

Usually transcriptional activators
Repressors are frequently found in bacterial systems, but a lot less often in eukaryotes as a
chromatin packaged genome is already in a repressed state

Many gene-specific TFs are only expressed in particular cell types

Work together to regulate the expression of a particular set of genes

Gene-specific TFs must be able to specifically bind a subset of genes through sequence-specific
DNA-binding domains. The also need to modulate the activity of promoter bound transcription
machinery (RNAP + basal factors) e.g. stimulate TFIIH to increase helicase activity.

Gene-specific TFs consist of a DNA binding domain (that is specifically folded for DNA binding)
and a number of activation domains.

DNA footprinting
1. DNA is end-labelled
2. DNA is treated with restriction enzymes
3. DNA is denatured to remove protein
4. Fragments are separated using PAGE
5. Bands are visualised by exposing gel to X-ray film
Allows mapping of TF binding sites

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Molecular Biology II Eukaryotic Gene-Specific Transcription Factors

Physical Chemistry of Transcription:

DNA and proteins interact in the following ways

 Electrostatic bonds
 Hydrogen bonds
 Van der Wall forces
 Hydrophobic interactions

Positive charges in proteins (Arg/Lys/His) will interact with the negatively charged phosphates in
the backbone of nucleic acids. The shape of a protein is important as well as charge.

Electrostatic interactions between proteins and the DNA backbone provide stability, but no
specificity.

Bases are

accessible in dsDNA without melting via the major (12Å wide) / minor (6Å) groove

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Molecular Biology II Eukaryotic Gene-Specific Transcription Factors

Points of recognition via the major / minor grooves

Absolute recognition of the 4 bases is possible via the major groove

TFs that bind the minor groove can only distinguish between A-T / T-A from G-C / C-G

DNA-Binding Domains:
All use the ‘α-helix in the major groove’ method

I) Helix-turn-helix motif (many variants)

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Molecular Biology II Eukaryotic Gene-Specific Transcription Factors

 Used in many bacterial TFs (e.g. lac repressor, CAP protein)

 Also found in many important eukaryotic TFs
 Contain 2 α-helices separated by a loop
o C-terminal helix (recognition helix) fits in major groove
o N-terminal helix stabilises structure and positions recognition helix in groove
 Q (glutamine) residues in the recognition helix make sequence specific contact
 HTH motifs are often found as dimers, and so recognise palindromic sequences
o e.g. λ phage repressor

II) Helix-loop-helix motif

 Several TFs involving cell proliferation contain HLH DNA binding domains
 The c-jun/c-fos heterodimer is a transcriptional activator known as AP-1
 Both c-jun & c-fos have been identified as oncogenes
o Can turn a normal cell into a cancerous one if mutation / overexpression occurs
 HLH motifs must dimerise before they can bind to DNA

III) Zinc-finger motifs

 Contain a zinc atom that is co-ordinated by Cys/His residues in a particular configuration
 Only found in eukaryotes
 Many 1000s proteins in humans are known to contain them
 The single helix goes into the major groove
 Usually work in tandem
 Each zinc-finger identifies a 3 nucleotide motif ( 1/64 triplets)
 Complex DNA sequences can be recognised by arranging different zinc-fingers in tandem
 3 zinc fingers provide a 9 nucleotide recognition sequence
o Useful for making artificial TFs

p53

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Molecular Biology II Eukaryotic Gene-Specific Transcription Factors

A tumour suppressor, which, in response to DNA damage slows progression through the cell cycle
and initiates apoptosis if damage is severe
Tumour-specific point mutations occur in many human cancers, of which around 50% contain a
p53 mutation
p53 is very complex, so its easy for something to go wrong
p53 acts as a TF and has a central domain which binds DNA in a sequence specific manner
This domain has no structural similarity to any other DNA binding protein
Most of the p53 mutations that bring about cancer are found in the DNA binding domain

Activation Domains:

Once a target site has been recognised by gene-specific TFs, the basal transcription machinery
has to be stimulated, which is carried out by activation domains.

Many are unstructured which means they cannot be crystallised. This makes analysis harder.

Can help to create ‘open’ chromatin domains

Lecture 20 [Page 5]